CN101805712B - Bacillus subtilis and application thereof in producing (R)-mandelic acid with biological catalysis method - Google Patents
Bacillus subtilis and application thereof in producing (R)-mandelic acid with biological catalysis method Download PDFInfo
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Abstract
The invention belongs to the biochemical industry field, which discloses a bacillus subtilis and application thereof in producing (R)-mandelic acid with biological catalysis method. Biological catalyst which is obtained by mixing the bacillus subtilis KR2 (CGMCC NO.3242) wet cells and covalent modifier is used in organic solvent to catalyze and hydrolyze 1 to 10 percent of mandelonitrile to the mandelic acid at 15 to 35 degrees centigrade and a speed of 100-400 rpm. The bacillus subtilis KR2 (CGMCC NO.3242) has strong tolerance towards the organic solvent, overcomes the defects of easy damage, low enzyme activity and the like for common microbiological cells in an organic solvent system and has good substrate stereoselectivity at the same time. The optical purity of the obtained (R)-mandelic acid reaches more than 95 percent e. e and the yield reaches more than 70 percent.
Description
Technical field
The invention belongs to biological chemical field, relate to the new bacterial strain of a strain---subtilis (Bacillus subtilis) KR2, and the application of this bacterial strain in biological catalysis production (R)-amygdalic acid.
Background technology
(R)-amygdalic acid claims Alpha-hydroxy toluylic acid, phenylglycollic acid, mandelic acid again, is soluble in ether, Virahol, is dissolved in the second alcohol and water.This compound is a kind of important chiral drug intermediate, is widely used in the synthetic of multiple optical activity medicine, as microbiotic such as semisynthetic penicillin and cephamycins, and slimming medicine, antitumor drug, agricultural chemicals and other drug.International market demand increases with average annual about 10% approximately at present, and market potential is huge.
Mandelonitrile claims mandelonitrile, Maleic nitrile etc. again, and light yellow transparent oily liquid is dissolved in ethanol, ether, chloroform, and is water-soluble hardly.Phenyl aldehyde is dissolved in the chloroform, makes mandelonitrile, get amygdalic acid through chemical method or biological catalysis hydrolysis again but add anhydrous prussic acid simple reaction.Wherein big with the chemical hydrolysis quantity of three wastes, water is seriously polluted, and yield is low, and the racemic mandelic acid form is present in the reaction beyond the product, need carry out complicated fractionation.Biological catalysis can adopt highly stereoselective nitrilase that racemic mandelonitrile one one-step hydrolysis is made to have optically active amygdalic acid isomer.
In addition, the preparation method of (the R)-amygdalic acid that has possessed at present mainly contains chromatography, chemical resolution method and biological catalysis.Characteristics such as chromatography has fast, and product purity height and method are easy, but need expensive equipment such as chiral column, treatment capacity is little, and the cost height is not suitable for large-scale production.Chemical resolution method mainly is to adopt chiral separation agent and two kinds of enantiomorphs of amygdalic acid to form diastereoisomeric salt, to increase its differences of physical properties so that Crystallization Separation.This method has the application potential of large-scale production, but shows limitations such as productive rate is low, the optical purity of product is not high, resolution reagent is more expensive, big for environment pollution.Biological catalysis mainly is to utilize the interior relevant enzyme of organism that the height stereoselectivity of substrate is carried out catalytic conversion reaction, product optical purity height, and the yield height, side reaction is few, can carry out safety and environmental protection at normal temperatures and pressures.Again multiple route selection can be arranged according to substrate is different with catalyzer (biological enzyme) kind, as: be substrate all among patent CN 1840671A and the CN1715398A, utilize the difference of two enantiomer degradation speeds in the external racemic mandelic acid of specified microorganisms to realize splitting with racemic amygdalic acid.The weak point of this method be with degraded wherein the mode of a certain enantiomorph obtain another kind and have optically active amygdalic acid, its ultimate yield is not higher than 50%, wastage of material is more.Reported all among CN 1580270A, CN 1840670A and the CN 1710087A that employing benzoyl formic acid or derivatives thereof as substrate, adopts different microorganisms to split the chiral mandelic acid who obtains single configuration respectively.But benzoyl formic acid belongs to alpha-ketoacid, oxidation easily, and ketone group is taken off in decarboxylation, gets relatively difficulty of system, and price is more expensive, and is higher relatively as amygdalic acid suitability for industrialized production raw materials cost with this.
Summary of the invention
The objective of the invention is at above the deficiencies in the prior art, the new genus bacillus of a strain (Bacillussubtilis) KR2 (CGMCC NO.3242) is provided; It is substrate with the mandelonitrile in the nonaqueous phase system that another object of the present invention provides this bacterium, the application in biocatalysis production (R)-amygdalic acid.The present invention is substrate with the mandelonitrile; adopt the nonaqueous phase system to carry out biocatalytic reaction; be intended to solve in the prior art low for product yield in substrate biocatalysis product (R)-amygdalic acid with the mandelonitrile; substrate separates crucial restriction such as difficulty technical barrier with product, be that large-scale production high purity (R)-amygdalic acid provides a feasible approach by the cost advantage of raw material.
Purpose of the present invention can reach by following measure:
Subtilis KR2 (CGMCC No.3242) has following microbial characteristic:
1. morphological feature
Circular slick single bacterium colony appears after cultivating 48 hours on the solid plate substratum, canescence, and opaque, surface wettability, the edge is smooth, whole bacterium colony projection, easily picking; It is shaft-like that microscopy becomes, size is long to be (0.5～0.8um) * (1.5～2.5um), give birth in the gemma.
2. cultivate and learn feature
This bacterial strain 30 ℃ of cultural characteristics of cultivating after 48～96 hours down in following 3 kinds of substratum see Table 1.
The cultural characteristic of table 1 subtilis KR2 (CGMCC No.3242) on 3 kinds of substratum
3. physiological and biochemical property
Table 2 subtilis KR2 (CGMCC No.3242) physiological and biochemical property
(annotate: "+" expression positive in the table, "-" expression is negative.)
The cultivation of subtilis KR2 (CGMCC NO.3242):
Carry out the preparation of subtilis KR2 (CGMCC NO.3242) inclined-plane seed earlier, cultivated 3～7 days down at 20～35 ℃ after subtilis KR2 (CGMCC No.3242) being inoculated into the inclined-plane of the bacterium of going out, scrape and get 3～5 ring bacterial classification inoculations in the triangular flask that sterilized liquid nutrient medium is housed, in temperature is 20～35 ℃, rotating speed is a shaking culture 30～120 hours under 100～300rpm, promptly can be used as subtilis KR2 (CGMCC No.3242) first order seed.Under the same culture condition, first order seed is equipped with in the triangular flask of same substratum by second batch of the inoculum size switching of 3～10% (%:v/v), similarity condition is cultivated after 30～72 hours as subtilis KR2 (CGMCC NO.3242) secondary seed down.Subtilis KR2 (CGMCCNO.3242) secondary seed is inoculated into the inoculum size of 3～10% (%:v/v) carries out fermentation culture in 7～300L fermentor tank, culture condition is: liquid amount: 60～70% (%:v/v), air flow: 0.1～1 (v/v.min), tank pressure: 0.02～0.05MPa, temperature: 20～35 ℃, rotating speed: 100～400rpm, fermentation time: 30～120 hours.
With the centrifugal 10min of fermented liquid 3000rpm in the fermentor tank, abandon supernatant after the fermentation ends, the centrifugal 10min of gained precipitate with deionized water resuspended back 3000rpm abandons supernatant, gets subtilis KR2 (CGMCC NO.3242) wet thallus.
Related medium component is as follows:
Slant medium (g/L): glucose: 10～30, yeast extract paste: 2～10, peptone: 2～10, sodium-chlor: 1～5, potassium primary phosphate: 0.5～3, dipotassium hydrogen phosphate: 0.5～3, sal epsom: 0.2～2, agar: 10～30, pH:5～9.
One-level, secondary liquid seed culture medium (g/L): glucose: 10～30, yeast extract paste: 2～10, peptone: 2～10, sodium-chlor: 1～5, potassium primary phosphate: 0.5～3, dipotassium hydrogen phosphate: 0.5～3, sal epsom: 0.2～2, hexanolactam: 0.5～10, isovaleronitrile: 0.1～5, cobalt chloride: 5～50ppm, pH:5～9.
Fermention medium (g/L): glucose: 10～50, yeast extract paste: 2～10, peptone: 2～10, sodium-chlor: 1～5, potassium primary phosphate: 0.5～3, dipotassium hydrogen phosphate: 0.5～3, sal epsom: 0.2～2, hexanolactam: 0.5～10, isovaleronitrile: 0.1～5, cobalt chloride: 5～50ppm, pH:5～9.
The application of subtilis (Bacillus subtilis) KR2 (CGMCC NO.3242) in synthetic (the R)-amygdalic acid of biological catalysis.
A kind of method of utilizing subtilis KR2 (CGMCC NO.3242) biological catalysis production (R)-amygdalic acid in the nonaqueous phase system.Fig. 1 is seen in the technical process of this method.
The concrete steps of this method are:
(1) preparation of biological catalyst
Subtilis KR2 (CGMCC NO.3242) wet thallus and the covalency modifier mass ratio with 1: 1～1: 50 is mixed, and this mixed solution promptly can be used as biological catalyst of the present invention.The polyoxyethylene glycol of molecular weight≤700 is selected in described covalent modification agent for use, and described subtilis KR2 (CGMCC NO.3242) wet thallus and covalency modifier mix with 1: 1～1: 5 mass ratio.
(2) nonaqueous phase catalyzed reaction
The biological catalyst for preparing is joined with 5～15% of liquid amount volume in the stirring tank that organic solvent is housed, and the add-on of organic solvent is 50～70% (v/v) of stirring tank volume, and the addition of mandelonitrile is 1～10% (g/100mL) of liquid amount.In temperature is 15～35 ℃, and mixing speed is to be amygdalic acid with the mandelonitrile catalytic hydrolysis under 100～400rpm.When trending towards zero, concentration of substrate can finish reaction.
Wherein said organic solvent is selected from any one in ethyl acetate, methylene dichloride, toluene, isopropyl ether, mibk, propyl carbinol, the Virahol, ethyl acetate.
Described catalyzer removes the prepared subtilis KR2 through covalent modification (CGMCC NO.3242) extracellular in the step (1); can also be free cell, immobilized cell particle or the flocculation enchylema of subtilis KR2 (CGMCC NO.3242), preferably through the subtilis KR2 of covalent modification (CGMCC NO.3242) cell.
(3) product separates
The reaction solution that ultrafiltration membrane system is filtered out concentrates 75～85 ℃ of distillations, and 0～4 ℃ of crystallization in washing back just gets amygdalic acid.Distillation and the organic phase of washing out all can continue to overlap to use down in batch catalyzed reaction goes.
Beneficial effect of the present invention:
The katalysis that the present invention mainly utilizes subtilis KR2 (CGMCCNO.3242) body endoenzyme is hydrolyzed into mandelonitrile (R)-amygdalic acid in the nonaqueous phase system.This subtilis KR2 (CGMCC NO.3242) is strong to the tolerance of organic solvent, has overcome the easy inactivation of common micro-organisms cell biological enzyme in organic solvent system, the enzyme deficiency such as low of living, and simultaneously substrate is shown stereoselectivity preferably.The optical purity of gained (R)-amygdalic acid reaches more than the 95%e.e, (R)-and the productive rate of amygdalic acid reaches more than 70%.Reaction solution is through concentrating, and after the washing, product (R)-amygdalic acid is dissolved into aqueous phase preferably, residual substrate mandelonitrile is soluble in the aqueous phase hardly, can use in the next batch reaction with the organic phase cover, realize that not only substrate separates preferably with product, and indirect raising substrate utilization ratio.
The preservation of the biological material specimens that relates to
The present invention subtilis required for protection (Bacillus subtilis) KR2 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; the address is a Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica; preserving number is CGMCC NO.3242, and preservation date is on August 19th, 2009.
Description of drawings
The process flow sheet of Fig. 1 subtilis KR2 (CGMCC NO.3242) biological catalysis production (R)-amygdalic acid in the nonaqueous phase system.
Embodiment
Embodiment 1 bacterial screening
Subtilis involved in the present invention (Bacillus subtilis) KR2 (CGMCC NO.3242) be the member of biochemical industry study group of our company from polluted by nitrile compounds to reach more than 15 years insecticide factory's soil screen and obtain.
The screening method of bacterial classification is as follows among the present invention:
Get surplus the mud sample 100 batch from Nanjing first insecticide factory's sewage lagoon different angles, laggard row filter of enrichment culture and evaluation in batches: take by weighing 10g mud, add sterilized water 50mL, be inoculated in the 45mL enrichment medium with getting the 5mL suspension liquid after the granulated glass sphere concassation, place on 30 ℃ of shaking tables 120rpm shaking culture 3 days.Get this nutrient solution afterwards and carry out gradient dilution, get 10
-6, 10
-7, 10
-8Gradient dilution liquid be applied on the flat board that contains the primary dcreening operation solid medium, put 30 ℃ of constant incubators and cultivated 3～6 days.Single colony inoculation that picking grows enlarged culturing to the new primary dcreening operation solid medium, culture condition is the same.Treating to scrape off after bacterium grows 3～5 rings is inoculated in the liquid fermentation medium and cultivates.Shaking culture is 3 days under 120rpm on 30 ℃ of shaking tables, gets centrifugal 10min under the 15mL nutrient solution 3000rpm, goes to precipitate thalline with isopyknic normal saline flushing after the supernatant liquor, continues the centrifugal thalline that gets.The gained thalline added in the PBS damping fluid that contains 1% (%:g/100mL) iminodiacetonitrile to transforming 2 hours, cell concentration is 0.5g/L in the reaction system.Get that centrifugal 10min gets supernatant liquor under the 5mL conversion fluid 3000rpm, the HPLC method is clear liquid analytically.Illustrate that this bacterium has conversion capability to iminodiacetonitrile if conversion fluid contains iminodiethanoic acid, select the wherein the highest strain of iminodiacetic acid conversion, by " simple and clear uncle's outstanding Bacteria Identification handbook the 8th edition is accredited as subtilis.With this bacillus subtilis called after KR2, be preserved in Chinese microbial preservation management committee common micro-organisms center (CGMCC), preserving number CGMCC NO.3242 undetermined, preservation date are on August 19th, 2009.
Subtilis KR2 among the present invention (CGMCC NO.3242) can be simultaneously respectively with vinyl cyanide, cigarette nitrile, lactonitrile, hydroxyacetonitrile, iminodiacetonitrile is to grow on the solid primary dcreening operation substratum of only nitrogen source, the substrate scope is wide, degraded nitrile compounds ability is strong, is suitable as the production application of iminodiethanoic acid among the present invention.
It is as follows respectively that described liquid enrichment medium in the screening method, solid primary dcreening operation substratum, liquid sieve substratum again:
Enrichment medium (g/100mL): glucose: 3, yeast extract paste: 0.5, peptone: 0.5, sodium-chlor: 0.1, potassium primary phosphate: 0.1, dipotassium hydrogen phosphate: 0.1, sal epsom: 0.05, pH:7,121 ℃ of sterilization 20min.
Solid primary dcreening operation substratum (g/100mL): glucose: 3, iminodiacetonitrile (or vinyl cyanide or cigarette nitrile or lactonitrile or hydroxyacetonitrile): 1, sodium-chlor: 0.1, potassium primary phosphate: 0.1, dipotassium hydrogen phosphate: 0.1, sal epsom: 0.05, agar: 2, pH:7,121 ℃ of sterilization 20min.
Liquid fermentation medium (g/100mL): glucose: 3, yeast extract paste: 0.5, peptone: 0.5, sodium-chlor: 0.1, potassium primary phosphate: 0.1, dipotassium hydrogen phosphate: 0.1, sal epsom: 0.05, urea: 0.5, pH:7,121 ℃ of sterilization 20min.
The preparation of embodiment 2 subtilis KR2 (CGMCC No.3242) biological catalyst
In aseptic Bechtop, scrape and get subtilis KR2 (CGMCC No.3242) slant strains 3 ring with transfering loop, be inoculated in the level liquid seed culture medium (glucose: 15, yeast extract paste: 5, peptone: 5, sodium-chlor: 2, potassium primary phosphate: 1, dipotassium hydrogen phosphate: 1, sal epsom: 0.5, hexanolactam: 5, isovaleronitrile: 1, cobalt chloride: 10ppm, pH:7.0, unit: g/L).In temperature is 25 ℃, rotating speed be under the 150rpm shaking culture got final product in 48 hours one-level subtilis KR2 (CGMCC No.3242) seed liquor, this primary seed solution is transferred to be equipped with proceeding in the sterilized substratum of sample ingredient according to 10% inoculum size cultivates, can get subtilis KR2 (CGMCC No.3242) secondary seed solution after 48 hours.
Subtilis KR2 (CGMCC No.3242) secondary seed solution is inoculated in the sterilized liquid fermentation medium of 35L according to 10% inoculum size, (glucose: 30, yeast extract paste: 10, peptone: 10, sodium-chlor: 2, potassium primary phosphate: 1, dipotassium hydrogen phosphate: 1, sal epsom: 0.5, isovaleronitrile: 1, hexanolactam: 5, cobalt chloride: 10ppm, pH:7, unit: g/L), the fermentor tank volume is 50L, adopt blowing air to control dissolved oxygen and be not less than 10% with the mode that stirs coupling, air flow: 0.2v/v.min, tank pressure: 0.03MPa, temperature: 25 ℃, initial rotating speed 100rpm, according to the adjusting in steps of dissolved oxygen of fermentation liquid level, maximum speed of revolution is no more than 400rpm in the fermenting process, ferments to make subtilis KR2 (CGMCC NO.3242) cell fermentation liquid after 72 hours.The centrifugal 10min of this fermented liquid 3000rpm gets sedimentation cell, continues the centrifugal 10min of 3000rpm with the resuspended back of deionized water, abandons supernatant and must precipitate wet thallus.
Subtilis KR2 (CGMCC NO.3242) wet thallus and polyoxyethylene glycol (molecular weight is 200) are mixed with 1: 3 mass ratio, and this mixed solution promptly can be used as biological catalyst of the present invention.
Embodiment 3 enzyme biopsies are surveyed
Present embodiment adopts the fermented liquid of subtilis (Bacillus subtilis) CGMCC NO.3242 to carry out the mensuration that enzyme is lived.The definition that enzyme is lived: under 25 ℃, the micrograms of the amygdalic acid that 1 gram wet thallus and 1 milliliter of mandelonitrile catalyzed reaction generated after 1 hour, this micrograms is total enzyme activity unit number.The HPLC method is adopted in the detection of amygdalic acid, and detect parameters is chromatographic column: LC-(R)-3,5-DNBPG chiral column, 150mm * 4.6mm I.D, moving phase: isopropanol (30/70), flow velocity: 0.5mL/min, column temperature: 25 ℃, detection: UV 230nm.Under condition of the present invention, biomass reaches as high as the 47mg wet thallus in every milliliter of fermented liquid, and total enzyme work reaches as high as 985U.
Embodiment 4 nonaqueous phase catalyzed reactions
In the 50L stirring tank, the 35L ethyl acetate is housed, add the 1.8kg mandelonitrile, average mark adds for 2 times, and the back that stirs adds the biological catalyst of preparation among the 3.5L embodiment 1.In temperature is 25 ℃, and mixing speed is a catalyzed reaction under the 150rpm, per hour measures concentration of substrate one time, finishes reaction when the concentration that detects mandelonitrile trends towards zero.
Embodiment 5 products separate
With the reaction solution among the embodiment 4 with the ultrafiltration membrance filter of 0.2um, afterwards 80 ℃ down distillation concentrate crude product, reclaim solvent simultaneously and apply mechanically.The crude product that obtains is washed with the deionized water of 5 times of volumes, and residual insoluble organic phase cover is used in the following batch of reactor.At 4 ℃ of following freezing and crystallizings, remaining liquid evaporation moisture continues crystallization with water, and getting optical purity at last is (R)-amygdalic acid 1.503kg of 96.6%e.e, and yield reaches 70.6%.
Embodiment 6
Present embodiment only changes the addition of catalyzer, the remaining reaction condition, and the substrate addition is fully consistent with embodiment 4, embodiment 5 with the product sepn process.Carry out two batch reactions separately, the addition of biological catalyst is respectively 2L and 5L in the reactor.At last respectively after 52 hours and 27 hours in the detection reaction liquid content of mandelonitrile be zero substantially, carry out product behind the stopped reaction and separate.Obtaining optical purity at last respectively is (R)-amygdalic acid 1.485kg of 97.1%e.e and (R)-amygdalic acid 1.512kg that optical purity is 97.5%e.e, and yield reaches 70.1% and 71.7% respectively.
Embodiment 7
Present embodiment only changes the addition of substrate mandelonitrile in the reactor, the remaining reaction condition, and catalyzer addition and product sepn process are consistent with embodiment 4, example 5 fully.The accumulation addition of mandelonitrile is 3kg in the present embodiment, divides 3 addings, and each addition is 1kg.Obtaining optical purity at last is (R)-amygdalic acid 2.631kg of 95.2%e.e, and yield reaches 73.1%.
Embodiment 8
Present embodiment only changes the temperature of catalyzed reaction, the remaining reaction condition, and the substrate addition is fully consistent with embodiment 4, embodiment 5 with the product sepn process.Control reaction temperature is 10 ℃, reacts termination reaction after 36 hours, and finally obtaining optical purity is (R)-amygdalic acid 1.55kg of 96.8%e.e, and yield reaches 72.9%.
Embodiment 9
Present embodiment is mainly examined or check the genetic stability of subtilis KR2 (CGMCC NO.3242).Take out after activating 20 minutes in 30 ℃ of constant incubators of this subtilis KR2 (CGMCC NO.3242) placement with preservation in 4 ℃ of refrigerators and be inoculated in the fresh slant medium, placing 30 ℃ of constant incubators afterwards cultivated 3 days, treat to grow on the inclined-plane first filial generation that is this subtilis behind the new line bacterium colony, called after KR2-1; Be the bacterial classification that sets out with KR2-1 afterwards, inoculate in the same way and go down to posterity, cultivate called after second filial KR2-2 behind the line bacterium colony that makes new advances; Be the bacterial classification that sets out with KR2-2 again, the new slant medium of transferring in the same way gets F3 KR2-3.Continuous passage is 30 times in this way, obtains KR2-4, KR2-5......KR2-30 respectively.Slant medium (g/L): glucose: 15, yeast extract paste: 5, peptone: 2, sodium-chlor: 1, potassium primary phosphate: 0.5, dipotassium hydrogen phosphate: 0.5, sal epsom: 0.5, agar: 20, pH:7.
Select resultant KR2-10, KR2-20, KR2-30 three strain filial generations in the above cultivation of going down to posterity, with original KR2 (CGMCC NO.3242) is contrast, carry out the preparation of level liquid seed, secondary liquid seeds simultaneously, working method and substratum all with embodiment 1 in identical.Carry out the preparation of free cell afterwards, the secondary liquid seeds of gained is inoculated into 10% inoculum size shaking in the bottle of sterilization fermentation substratum is housed, bottle final liquid amount that shakes of every 500mL capacity is 100mL, laying temperature is a shaking culture 3 days on 25 ℃ the constant temperature shaking table after having connect kind, and rotating speed is 150rpm.Identical among described fermention medium and the embodiment 1.
With the liquid fermentation liquid of above gained frozen centrifugation 10 minutes under 3000rpm, abandon after the supernatant liquor with behind isopyknic deionized water rinsing precipitation thalline one time and continued 3000rpm centrifugal 10 minutes, abandon supernatant, wet thallus.The precipitation wet thallus and the polyoxyethylene glycol (molecular weight is 200) of gained are mixed with 1: 3 mass ratio, add form as biological catalyst of the present invention.
In the 500mL flask, add the 300mL ethyl acetate, the KR2 for preparing more than the 30mL (KR2-10, KR2-20, KR2-30) biological catalyst, 10g mandelonitrile.In temperature is 25 ℃, and mixing speed is a catalyzed reaction 30 minutes under the 150rpm, and sampling detects behind the 0.2um membrane filtration afterwards.The concentration of amygdalic acid is respectively 1.25%, 1.23%, 1.20%, 1.18% (%:g/100mL) in KR2, KR2-10, KR2-20, the pairing conversion fluid of KR2-30 four strain bacterium as a result.Above result as seen, this subtilis KR2 (CGMCC NO.3242) still keeps and the almost suitable transformation efficiency of parental generation KR2 bacterium after 30 times going down to posterity, and has good genetic stability.
Claims (3)
1. preserving number is the application of subtilis KR2 in synthetic (the R)-amygdalic acid of biological catalysis of CGMCC No.3242.
2. application according to claim 1 is characterized in that a kind of concrete steps of subtilis KR2 synthetic (R)-amygdalic acid method of biocatalysis in the nonaqueous phase system of utilizing are:
(1) preparation of biological catalyst
The polyoxyethylene glycol of subtilis KR2 wet thallus and molecular weight≤700 mass ratio with 1: 1～1: 50 is mixed, and this mixed solution is as biological catalyst moiety of the present invention;
(2) nonaqueous phase catalyzed reaction
Join the biological catalyst for preparing in the stirring tank that ethyl acetate is housed with 5～15% of liquid amount volume, the add-on of ethyl acetate is 50～70% of a stirring tank volume, the addition of mandelonitrile is 1～10% of a liquid amount, in temperature is 15～35 ℃, mixing speed is to be amygdalic acid with the mandelonitrile catalytic hydrolysis under 100～400rpm, can finish reaction when concentration of substrate trends towards zero;
(3) product separates
With reaction solution behind 0.2～0.5 μ m membrane ultrafiltration in 75～85 ℃ of evaporation concentration, 0～4 ℃ of crystallization in washing back gets amygdalic acid.
3. application according to claim 2, the mass ratio that it is characterized in that the polyoxyethylene glycol of described subtilis KR2 wet thallus and molecular weight≤700 is 1: 1～1: 5.
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